We have developed data in a murine transplantation model which indicates that it is not necessary to open marrow spaces for effective marrow engraftment. Evaluating transplantation of male BALB/c marrow cells into non-myeloablated female BALB/c mice we have also shown that marrow harvested six days after 5-fluorouracil (5FU) is defective in engraftment and finally that engraftment seen in this model is stable out to two years post-transplantation. More recently, we have developed information indicating that the relatively high engraftment (over 70% in some cases) rates are dependent on the schedule of transplantation, (i.e., giving the same number of cells divided into more frequent infusions gives better engraftment). We have also generated data suggesting that the engraftment defect seen with post-5FU marrow is transient and may be due to cycle induction. Further, the normal male marrow cells engrafting into normal female hosts appear to not be in active cell cycle and to be lineage negative and Ly6 (SCA) positive. The present proposals plans to extend these results establishing the optimum schedule and cell dose for engraftment into non-myeloablated hosts and to evaluate non male-female transplant models (a beta-galactosidase transgenic ROSA261SvJ129 model or a Ly5.1/Ly5.2 congenic model). We plan to study whether host stem cells are displaced or augmented in this model, and whether the engraftment defect seen after the treatment of mice with 5FU or exposure of cells to cytokines is related to the cycle status of the stem cell. We will also attempt to identify cytokines which may be able to maintain marrow engrafting cells without inducing progression through the cell cycle. Lastly, we plan to continue to study the phenotype of engrafting cells and the ability of purified stem cells to engraft in this model. We will utilize liquid and clonal agar cultures, in vivo transplantation of marrow into mice by tail vein injection, Southern blot analysis and fluorescence in situ hybridization (FISH) to identify male DNA or cells, fluorescent activated cell sorting (FACS) with immunomagnetic bead separations to purify stem cells, and standard histologic techniques to assess marrow cellularity. These studies should establish a new transplant approach and characterize a different marrow stem cell. This particular transplantation approach will be particularly useful for approaches to gene therapy for HIV infected individuals or individuals with sickle cell anemia or thalassemia.